Identification of anthocyanins in deep colored berries and grains in China

Identification of anthocyanin relies heavily on authentic anthocyanin standards and the detection instruments employed, and both of these made tremendous improvement in the past decades. Here, with 118 authentic anthocyanin standards and state-of-the-art detection method, we comprehensively analyze the anthocyanin composition of the most commonly seen, deep-colored berries and grains in China. Cyanidin-3-O-glucoside is the overwhelmingly major anthocyanin in all the berries and grains examined, including blue honeysuckle, blueberry, cranberry, elderberry, mulberry, black rice, and black soybean, which accounts for an average of 82% of the total anthocyanins with a little variation between them. Peonidin-3-O-glucoside is the second most abundant anthocyanin ranging from 2.6%–14.9% of the total anthocyanins. Pelargonidin-3,5-O-diglucoside is only found in blue honeysuckle, and besides that, berries and grains share a dominant portion of common anthocyanins among them. This study primes the survey of anthocyanin in common Chinese foods for the establishment of a nutrition database.


Introduction
Anthocyanins are chemicals composed of an aglycone (the anthocyanidin), sugar(s) and usually acyl group(s) which exhibit multiple health benefit to humans (Liang, Liang, Guo, & Yang, 2021).Anthocyanins are structurally diverse due to not only different hydroxyl and/or methoxyl substitutions at the R1, R2, R3 positions of the flavylium (2phenylbenzopyrilium) structure, but also different sugar groups attached (Liang et al., 2021).Subsequently, their diverse structures bring about different functions.For example, cyanidin-glucoside could inhibit Aβ aggregation and may help to enhance the cognitive function of mice via synergistically functioning with other anthocyanins(N.Y. Kim & Lee, 2016;Wong, Musgrave, Harvey, & Smid, 2013).On the other hand, cyanidin-sophoroside which carries different sugar group, exhibits antioxidative activity (Proteggente et al., 2002).Peonidin-glucoside, which differs from cyanidin-glucoside from the substitution at the R1 position, improves plasma antioxidant capacity, inhibits nitric oxide production in macrophages and suppresses reactive oxygen species (Hu, Zawistowski, Ling, & Kitts, 2003;Ramirez-Tortosa et al., 2001;Wang & Mazza, 2002).Thus, it is meaningful to fully utilize anthocyanins from food and agricultural byproducts (Zhou & Yang, 2022).Currently, it is difficult to obtain commercial products with anthocyanins as the main functioning components and dietary is still the main source of anthocyanin intake worldwide (Zhang et al., 2021).
Currently, consumers are intuitively informed that colored foods are often beneficial to their health, and some of them know that it is partially because of the anthocyanins rich in these foods.However, little is known about the variety and content of anthocyanins a specific food contains.This jeopardizes the intake of specific anthocyanin for the purpose of precision nutrition.For this, the Nutrient Data Laboratory of the United States Department of Agriculture established a flavonoid database (FDB) in 2003 and a proanthocyanidin database (PDB) in 2004 (Harnly et al., 2006).Survey of anthocyanin contents in common foods was executed with valid national sampling plan and analytical methods in the United States (Wu et al., 2006;Wu & Prior, 2005).Occasionally, the anthocyanin content of a local anthocyanin-rich food is also reported for reference elsewhere in the world.For example, the anthocyanin contents of wild blueberries in Québec area were reported previously (Nicoue, Savard, & Belkacemi, 2007).However, there is currently no data base of anthocyanin distribution in the common foods in China.
Thus, here we determine the anthocyanin compositions of the most colored berries seen in Chinese markets, including blueberry, blue honeysuckle (Lonicera caerulea L.), cranberry, elderberry, mulberry, and the most colored grains commonly available to Chinese citizens, including black rice and black soybean.All of these berries and grains are reported exhibiting antioxidative activity associated with their anthocyanin contents (Domínguez et al., 2020;Felgus-Lavefve, Howard, Adams, & Baum, 2022;I. Kim & Lee, 2020;Kumar et al., 2023;Wang et al., 2017;Zhang et al., 2023;Zhang, Zhang, Zhang, & Liu, 2010).This study aims to comprehensively analyze almost all possible anthocyanin components in the commonly consumed, deep colored berries and grains, to lay a foundation for the precise intaking of dietary anthocyanins.

Materials
Berries (including cranberry, elderberry, and mulberry) and grains (including black rice and black soybean) were purchased from Shaanxi Evergreen Biotech.Ltd. (Xi'an, Shaanxi, China).And these above berries and grains were collected in Shaanxi Province in 2023.Blueberry was collected from the Fenglin County, Heilongjiang Province in May 2023.Blue honeysuckle (cultivar of Lanjingling) collected in June 2023 was kindly gifted from Prof. Junwei Huo at College of Horticulture and Landscape Architecture, Northeast Agricultural University, Harbin, Heilongjiang Province.
Chemicals including absolute ethanol and acetic acid were purchased from Modern Oriental Fine Chemistry (Beijing, China), formic acid was purchased from Sigma-Aldrich (St. Louis, USA), hydrochloric acid was purchased from Sinopharm Chemical Reagent Co., ltd (Beijing, China), methanol was purchased from Merck (NJ, USA), potassium chloride and sodium acetate were purchased from Xilong Scientific (Shantou, Guangdong Province, China).

Anthocyanin extraction
Anthocyanins were extracted from the above berries and grains principally the same as described previously with minor modifications (Zhou & Yang, 2022).Briefly, berries and grains were mixed with 79% ethanol of pH 7.0 at a material liquid ratio of 1:15, smashed, and incubated at 64 • C for 50 min with the application of a VGT-1730QTD ultrasonic water bath (GT Sonic, Meizhou, Guangdong Province, China).The extract solution was centrifuged at 4000 rpm for 15 min at room temperature to remove precipitates, and filtered with a 0.45 μM PTFE microfiltration membrane.

Anthocyanin analysis
The above extracted anthocyanins were 10-folds diluted into a solution containing 50% methanol and 0.1% hydrochloric acid, vortexed for 5 min, sonicated for 5 min and centrifuged at 12000 r/min for 3 min.
On a ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 mm × 100 mm) equilibrated with a buffer containing 0.1% formic acid, 2 μL anthocyanin solution was loaded and eluted with increasing concentration of another methanol solution containing 0.1% formic acid.The flow rate was set at 0.35 mL/min and column temperature was set at 40 • C. The eluted anthocyanins were electrospray ionized and detected by their declustering potential and collision energy.
A total of 118 different authentic anthocyanin/proanthocyanin standards were applied to the above detection method.Among them, 46 were detected and a standard curve was established for each (Table 1) with concentrations of 0.01 ng/mL, 0.05 ng/mL, 0.1 ng/mL, 0.5 ng/mL, 1 ng/mL, 5 ng/mL, 10 ng/mL, 50 ng/mL, 100 ng/mL, 500 ng/mL, 1000 ng/mL, 2000 ng/mL, 5000 ng/mL loaded on the UPLC-MS/MS to establish the standard curve.Sample concentration was the input variable and corresponding peak area was the output.

Establishment of an exhaustive detection method
The UPLC-MS/MS detection method is currently the most widely used one to detect anthocyanins and its detection capacity mainly depends on the standard chemicals used.In our previous identification of anthocyanins from the grape pomace, only 15 authentic standards were used and thus the maximum number of anthocyanin species we could detect was limited (Zhou & Yang, 2022).Among them, the sugars are limited to glucoside, acetylglucoside, and coumaroylglucoside, and the anthocyanidins are limited to cyanidin, peonidin, delphinidin, petunidin, and malvidin.
As shown in Fig. 1, there are numerous peaks in the particle flow diagram generated by the MRM mode with the MS/MS instrument, indicating a mixture of different anthocyanins in each of the berry and grain samples analyzed.Since our detection mode allows quantitative analysis of anthocyanins, it is obvious that in each sample there is a couple of major anthocyanin components and several minor anthocyanin components.Each component and its proportion is identified and discussed in the following.

Major and minor anthocyanin components in berries and grains
Multiple peaks are detected for all of the 7 samples studied here, and apparently some anthocyanins are overwhelmingly richer than others.The identification and proportion of anthocyanins are quantitatively analyzed, and found that the distribution of anthocyanin in all the samples are highly heterogenous.To better analyze and describe such unevenly distributed anthocyanins, we define that anthocyanins accounting for more than 1% of the total anthocyanins are major components, and these accounting for less than 1% of the total anthocyanins are minor ones.
As for the minor anthocyanins, cyanidin-3-O-xyloside is present in all the samples examined here (Fig. 3).Cyanidin-3-O-(6-O-malonyl-β-Dglucoside), cyanidin-3,5-O-diglucoside and petunidin-3-O-glucoside are present among all the samples except blue honeysuckle.Quercetin-3-Oglucoside is present among all the samples except in mulberry.Pelargonidin-3,5-O-diglucoside is only found in blue honeysuckle, accounting for 985.6 ppm of the total anthocyanins, and not found in any other samples (Fig. 3a).There are no other specific anthocyanins exclusively present in other samples (Fig. 3b-g).Interestingly, there are more anthocyanins evenly distributed in the minor anthocyanin group of black soybeans than in other samples (Fig. 3g).

Common anthocyanin components in berries and grains
The common anthocyanins in berries and grains are analyzed with Venn diagram.All the 5 berries share a core of 11 common  Anthocyanin detection largely depends on the authentic anthocyanin standards used and no anthocyanins beyond the standards could be possibly detected.At the infant age of anthocyanin detection, there was often one or couple of anthocyanins identified due to the lack of authentic standards (Ashtakala & Maloney, 1971;Ng & Thimann, 1962).

Fig. 1 .
Fig. 1.Particle flow diagram of anthocyanin components in common berries and grains.The Multiple Reaction Monitoring mode of MS/MS detection exhibited multiple peaks, major and minor, indicating a mixture of anthocyanins found in each of the berry and grain extraction.

Fig. 2 .Fig. 3 .
Fig. 2. The major anthocyanin components of berries and grains detected.Anthocyanins accounting for more than 1% of the total anthocyanins are defined as major anthocyanins, and their distribution in different berries and grains are shown here as (a) blue honeysuckle, (b) cranberry, (c) blueberry, (d) elderberry, (e) mulberry, (f) black rice, and (g) black soybean.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4 .
Fig. 4. Venn diagram of common anthocyanins.(a) Venn diagram of anthocyanins between five berry samples examined.Blue indicates blueberry, orange indicates mulberry, dark blue indicates blue honeysuckle, purple indicates elderberry, and red indicates cranberry.(b) Venn diagram of anthocyanins between black rice and black soybean.Purple indicates black rice and grey indicates black soybean.(c) Venn diagram of anthocyanins between berries and grains.Green indicates berries and brown indicates grains.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 1
Standard curves of authentic anthocyanins.